Zeaxanthin, or 3,3'-dihydroxy-beta-carotene, is a natural carotenoid alcohol useful in the pigmentation of foodstuffs and cosmetics. For example, zeaxanthin imparts a desirable yellow to reddish-yellow color to the flesh, skin, and eggs of poultry and fish. Zeaxanthin is more desirable as a food dye than are chemically synthesized dyes, many of which have been banned by governmental agencies due to their mutagenic and carcinogenic potential. Natural carotenoids, which apparently have no toxic effects even at high concentrations, may replace yellow and red azo dyes that have proven to be harmful to animals.
Some carotenoids, such as zeaxanthin, are effective antioxidants due to their conjugated diene chemical structures. Thus, zeaxanthin may also be used to reduce damage to tissues caused by oxygen-free radicals, to prevent certain types of cancer, and to stabilize compounds subject to oxidation, particularly when exposed to light.
Zeaxanthin is preferred over other carotenoids for enhancing pigmentation in poultry and fish due to its potency, ability to provide a true color, and ability to deposit evenly in flesh and eggs. Zeaxanthin appears to be at least 1.5 times as potent as lutein. When administered to poultry in high doses, currently used carotenoid or carotenoid-containing compounds, such as canthaxanthin, alfalfa and cayenne pepper, cause abnormal red or purple colors in the flesh and color striations in yolks. High doses of lutein in feed has been shown to impart a greenish hue to poultry flesh and egg yolks. Beta-carotene does not deposit well in the flesh of poultry, and canthaxanthin apparently deposits in the iris of the eye. Zeaxanthin, in contrast, imparts a yellow-red color even at high doses and deposits well in poultry flesh, based on high zeaxanthin-containing corn feed studies. In addition, at least some fish and crustaceans, such as shrimp, goldfish, and carp, apparently can convert zeaxanthin into the red-colored pigment astaxanthin, suggesting that feeding of zeaxanthin to such fish and crustaceans will enhance desirable red coloration.
Zeaxanthin is produced along with other carotenoids in certain plants, such as corn, marigolds, and alfalfa. Although farmers have fed these plants to poultry, such diet supplementation is impractical due to the expense and lack of consistency in the amounts of zeaxanthin produced by such plants.
Prior attempts to purify zeaxanthin from natural sources have suffered from low yields. Moreover, the costs involved in chemically synthesizing zeaxanthin using existing techniques have proven to be prohibitive. In addition, due to concern over the health risks of compounds produced synthetically, there is a preference to produce food additives naturally, such as by microbial biosynthesis.
The Official Publication of the American Association of Feed Control Officials provides a list of microorganisms that the U.S. Food and Drug Administration (FDA) considers to be "generally recognized as safe" (GRAS) for use as food additives and for use in the production of food additives. Concerns regarding government approval for food additives make it desirable to produce zeaxanthin using GRAS microorganisms. However, the present inventors are not aware of any GRAS microorganism capable of producing sufficient amounts of zeaxanthin for use in a commercial process.
Among FDA-approved GRAS strains is NeoSpongiococcum, the only alga presently designated as GRAS for feeding to poultry to enhance yellow pigmentation (21 C.F.R. .sctn.73,275). Several investigators have attempted to use Neospongiococcum and other algal microorganisms to synthesize carotenoids, but none have succeeded in isolating a microorganism capable of producing commercial amounts of zeaxanthin. In fact, investigators have simply reported that algae produce low quantities of total xanthophylls, of which zeaxanthin is just a component. (Xanthophylls are oxygen-containing carotenoids such as astaxanthin, canthaxanthin, cryptoxanthin, lutein, rhodoxanthin, torulene, and violaxanthin, in addition to zeaxanthin.) For example, U.S. Pat. No. 3,108,402 by Kathrein, issued Oct. 29, 1963, discloses the use of a Neospongiococcum microorganism to produce up to 300 milligrams (mg) of total xanthophylls per liter of fermentation medium.
Similarly, U.S. Pat. No. 2,949,700 by Kathrein, issued Aug. 23, 1960, discloses the use of non-GRAS Chlorella and Chlorococcum algae to produce up to about 180 mg xanthophyll per liter of fermentation medium.
Investigators have described the production of zeaxanthin by several non-GRAS bacteria, fungi, and sponges, but none of these procedures have been commercialized. See, e.g., U.S. Pat. No. 2,974,044 by Farrow et al., issued Mar. 7, 1961; U.S. Pat. No. 3,841,967 by Dasek et al., issued Oct. 15, 1974; U.S. Pat. No. 3,891,504 by Schocher et al., issued Jun. 24, 1975; U.S. Pat. No. 3,951,742 by Shepherd et al., issued Apr. 20, 1976; U.S. Pat. No. 3,951,743 by Shepherd et al., issued Apr. 20, 1976; European Patent Publication No. 0393690 by Misawa et al., published Oct. 24, 1990; and PCT International Publication No. WO 91/03571 by Gierhart, published Mar. 21, 1991.